Sequence-specific targeting of endogenous nucleic acids can be a tool for example in the regulation of gene expression, sequence-specific mutagenesis, gene reprogramming, gene labeling, gene isolation and/or gene modification.
One class of oligonucleotides used for sequence-specific nucleic acid targeting are triplex-forming oligonucleotides (TFO). These oligonucleotides can form a triple-stranded helix (triplex) with the target nucleic acids via Hoogsteen or reverse-Hoogsteen interactions, with purines in the target, without disrupting the hydrogen bonds between strands in the target duplex. The triplexes, when localized downstream of the promoter (or the origin of replication) generally prevent transcription (or replication) of the target sequence. Thus, these triplex-forming oligonucleotides have been explored as tools for regulation of gene expression (for review see Helene and Toulme, Biochem. Biophys. Acta 1049:99 (1990)). TFOs also have been used for site-directed mutagenesis (Wang et al., Science 271:802 (1996)), gene isolation (Cantor et al., U.S. Pat. No. 5,482,836) and site-specific DNA cleavage (Strobel and Dervan, Science 249:73 (1990)).
However, there are severe limitations to the utility of TFOs because of the sequence dependence of triplex formation. For all known TFOs (both with natural and artificial bases and backbones) the target must comprise homopurine-homopyrimidine strands (i.e. the Watson strand is solely purines and the Crick strand is solely pyrimidines), with some small variations allowed (for review, see Frank-Kamenetskii and Mirkin, Annu. Rev. Biochem. 64:65 (1995)). This severely limits the applicability of these techniques.
In contrast, there are recombination enzymes (for example, the RecA family of recombinases) which can form nucleoprotein filaments with any oligonucleotide, and can subsequently target any selected sequence. These nucleoprotein filaments presumably disrupt the hydrogen bonds between the strands in the target duplex, and form stable sequence-specific complexes with one or both of these strands primarily via Watson-Crick interactions (though the presence of some additional interactions between nucleic acids within the complex has not been ruled out). (For review see Radding, Homologous Pairing and Strand Exchange Promoted by E. coli RecA Protein, in Genetic Recombination, American Society for Microbiology, pp193-230, 1988; and Kowalczykowski and Eggleston, Annu. Rev. Biochem. 63:991-1043 (1994)).
The additional advantage of the nucleoprotein filament over TFOs is the fact that these nucleoprotein filaments exhibit far more rapid initiation of the complex formation, i.e. the formation with the target sequence. For example, for RecA-covered filaments the on-rate constant of the first bimolecular step of the reaction is about the same as for Watson-Crick duplex formation (Bazemore et al., J. Biol. Chem. 272:14672 (1997)). This is one to three orders of magnitude larger than the rate for triplex formation (Rougee et al., Biochem. 31:9269 (1992)). This suggests that these filaments can be used in significantly smaller concentrations than the TFOs to achieve the same effect. Similarly, nucleoprotein filaments have been used for RecA-assisted restriction endonuclease (RARE) cutting of chromosomes (Ferrin and Camerini-Otero, Science 254:1494 (1991)).
However, for relatively short oligonucleotides, these kinds of complexes usually dissociate very rapidly after the RecA is removed, unless the target is strongly negatively supercoiled (which is unlikely to be the case for many eukaryotic targets, which are globally relaxed). Since deproteinization of the structure can occur spontaneously in living cells, the stability of these structures after deproteinization varies. In addition, deproteinized complexes are expected to be more convenient for some manipulations with DNA in vitro.
The ability to selectively inhibit the growth of a subset of cells in a mixture of cells has many applications both in culture and in vivo. Where two sets of cells have distinguishing characteristics, such as tumor cells which require expression of one or more genes, which are not expressed in normal cells or only expressed at a low level, there is substantial interest in being able to selectively inhibit the proliferation of the tumor cells. Where groups of cells are differentiating, and at one level of differentiation, expression of a particular gene is required, the ability to inhibit the expression of that gene can be of interest. Where cells are infected by viruses, parasites or mycoplasmas, the selective ability to inhibit the growth of the infectious agent can be an important goal.
In the studies of metabolic processes, differentiation, activation, and the like, there are many situations where it is desirable to be able to selectively increase or decrease the transcription of a particular gene. In this way, one can study the effect of a modulation in the transcription of the gene and expression of the gene product on the phenotype of the cell. In the extensive efforts to understand embryonic and fetal development, to define segmental polarity genes and their function, there is also interest in being able to selectively inhibit particular genes during various phases of the development of the fetus.
As in the case of the studies in culture, selective inhibition of particular genes can also be of interest in vivo. In many situations, cellular proliferation can be injurious to the host. The proliferation can be as a result of neoplasia, inflammation, or other process where increased number of cells has an adverse effect upon the health of the host.
There is, therefore, substantial interest in finding techniques and reagents which allow for selective modulation of particular genes, families of genes, and their associated regulatory sequences, so as to control intracellular molecular processes. Thus it is an object of the invention to provide novel compositions of nucleoprotein filaments that can be used in methods of regulating gene expression in a sequence specific manner. These methods and compositions also have applications in gene isolation, labelling, mutagenesis, modification, and in vitro manipulation of nucleic acids.